1. Field of the Invention
This invention relates to a method and apparatus for preparing biological tissue samples for ultrastructural analysis by avoiding significant modification of the ultrastructure of tissue during preparation of the samples themselves. It is well known in the medical arts that to examine tissue samples, and determine the cellular structure and function thereof, the tissue must be "fixed" prior to the application of nearly all analytical methodologies.
Although the examination of tissue samples by use of various microscopes or related magnifying apparatus has been practiced for many years, there has been an inherent problem in preparing tissue samples for use with contemporary high resolution analytical microscopes, such as the STEM electron microscopes, which permit the examination of sample constituents via X-ray analysis at powers of from 500.times. to 500,000.times. with point to point resolution of 2 to 3 Angstrom units.
Specifically, it is difficult to interpret the results of tissue analysis while concommitantly assessing the extent of various artifacts produced during the tissue preparation processes. It is thus essential that artifacts be avoided wherever possible. Another problem results from physical shrinkage of the tissue sample itself when subjected to the extreme but necessary for successful preparation procedures extent in current dogma. In most normal tissue preparation steps, tissue shrinkage is in the order of 40%-50%. This shrinkage inevitably results in alteration of ultrastructure and massive rearrangement of infrastructural resolution. The net result of this ultrastructural translation damage is inaccurate detail in descriptions via this analytical procedure.
During the so-called "Golden Age of Morphology" the predominant underlying goal in qualitative and quantitative microscopy has been an aesthetically pleasing image. This goal is readily attainable with the fixation methods and apparatus which are currently available. However, it has become essential that the aesthetically pleasing image, which is produced by the preparation process, also yields a tissue sample which accurately reflects the true condition of tissue in the living organism i.e., approaching the "living state". This is the problem which the process and apparatus of this invention address and solve. Magnification apparatus which is currently available for analytical use is technically more advanced than are current tissue preparation technicques which have been previously employed. The method of this invention results in the preparation of tissue samples which are readily usable on known magnification and analytical apparatus.
2. The Prior Art
The most common prior art method for preparation of tissue samples for analysis is by means of chemical fixation and organic solvent dehydration. Inherent in this process is the concommitant artifact creation, sample shrinkage and resultant damage to and modification of tissue characteristics. These modifications, whether in the form of artifacts or the like, require interpretation by the individual or apparatus analyzing or evaluating the sample. This introduces, in many instances, an unsatisfactory risk of error. Chemical fixation is a well known technique and has served the analytical biologist well for many years and undoubtedly will continue to do so in certain limited applications. However, as the use of tissue sample analysis becomes more complex and the use of such analysis becomes more widespread, alternatives to chemical fixation are demanded. This is especially true as advances are being made in the magnification and analytical apparatus which is available. It is necessary that tissue preparation methods and the apparatus necessary to prepare tissue samples be equally advanced as the analytical tools, i.e., electron microscopes, which are being used to analyze the samples. Obviously, if the technology for tissue sample preparation is behind the technology of microscopy then the advanced microscopes serve no purpose to the morphologist or other tissue examiner.
The most common alternative to chemical fixation and organic solvent dehydration is freeze drying cryofixed samples. Freeze-drying following cryofixation is a well documented and well known technique for tissue preservation. It has several advantages. Cryofixation freezedrying results in a near-instantaneous arrest of cellular metabolism. There is also a stabilization and retention of soluble cell constituents through elimination of solvent contact with the sample. These are significant advantages to cryofixation-freeze-drying that have resulted in a great deal of research endeavors in attempting to apply cryofixation and freeze-drying techniques to known tissue preparation processes.
Unfortunately, the use of freeze-drying has resulted in the identification of a number of disadvantages. The primary disadvantage in currently available freeze-drying techniques is prevention of the formation of ice crystals. As can be readily appreciated, the formation of ice crystals destroys the ultrastructural morphological characteristics of the tissue sample being reviewed. The image is distorted when the cytoplasm becomes reticulated. The formation of ice crystals in the sample can also result in a change in pH of the tissue (eutectic formation) which possibly can result in abnormal cross-linking of macromolecules. There is also the possibility that proteins will denature and precipitate. These are but a few of the disadvantages which are inherent in the freeze drying process.
This general topic is discussed in some detail together with other prior art methods in an article entitled "Freezing and Drying of Biological Tissues for Electron Microscopy", Louis Terracio and Karl G. Schwabe, published in The Journal of Histochemistry and Cytochemistry, Volume 29, No. 9 at pp. 1021-1028 (1981). Problems associated with artifact formation are described in "Understanding the Artifact Problem in Freeze-Fracture Replication: A Review", The Royal Microscopial Society, (1982) at 103-123.
General principles which have been found applicable to freezing techniques which have demonstrated utility in the preparation of tissue samples are that as the cooling rate increases, tissue fluids can be frozen without the separation of water to extracellular spaces. It has been found, however, that regardless of the rate of cooling, ice crystals are probably still formed but as the cooling rates increase the size of the intracellular ice crystals decreases. The small size of ice crystals at high freeze rates is of course a substantial advantage in morphological examination as this results in minimal artifact creation and minimal ultrastructural damage during dehydration.
Historically, the criterion by which the techniques for rapid freezing have been judged was not the cooling rate of the system but simply the temperature of the environment in which the tissue was frozen. Thus, the term rapid freezing has been applied to any system in which the cooling agent has a temperature of -150 degrees C. or below. The effectiveness of a cooling system is dependent upon the rate at which heat is removed from the sample. Heat transfer is dependent not only on the temperature of the freezing system but also on its physical and thermal characteristics, as well as the size and thermal characteristics of the sample.
The most commonly used technique for rapid freezing is to immerse or "quench" the sample in a fluid cooling bath. The most commonly used fluids for quenching are liquid nitrogen, isopentane, propane and fluorocarbons such as Freon 12 and Freon 22. Although liquid nitrogen is generally regarded as an ideal quenching fluid due to its low temperature (-196 degrees C.), there are inherent disadvantages in the use of liquid nitrogen due to the occurrence of tissue surface film boiling due to the low heat of vaporization of liquid nitrogen. Film boiling is a characteristic of liquid nitrogen that inhibits the heat transfer rates by actually insulating the sample.
An alternate prior method for rapid freezing is freezing on the surface of a chilled metal. This typically involves opposing the tissue sample to a polished flat metal surface by pressing it firmly against this surface of the metal. Silver and copper are typically used as the polished metal blocks. This method is designed to take advantage of the high thermal conductivities and heat capacities of these metals when cooled to liquid nitrogen or liquid helium temperatures. The critical step in chilling on the surface of a metal is making firm contact with the dry, chilled metal surface with no rotational, translational or rebounding motion. Certain commercially available apparatus having known utility in the medical arts address and provide "bouncefree" freezing. Credit for the development of this apparatus is generally accorded to Dr. Alan Boyne of the University of Maryland School of Medicine.
There has recently been verification that there is a direct correlation between cooling rate and ultrastructural preservation in quenching fluids. As the freezing rate increases over the range of 100-4100 degrees C. per second (liquid nitrogen - propane), there is a corresponding decrease in the size of ice crystals present and thus an improvement in morphological preservation.
The critical step in the subsequent tissue preparation process is invariably the sublimation - dehydration of the supercooled tissue fluids recently described as an adiabatic "molecular distillation" process. Once the appropriate supercooling method has been chosen and implemented, it is necessary to further process the tissue for microscopic evaluation, since electron microscopes or other magnification apparatus that allow the viewing of frozen hydrated specimens are not readily available. Thus, dehydration is an essential step in the preparation of biological tissue samples and a step which oftentimes results in the destruction via reticulation of the infrastructure and ultrastructure of the tissue morphology.
In certain prior drying techniques, the tissue sample had not been entirely solidified due to eutectic formation as the cellular fluid solutes are concentrated in bound water compartments. This transfer of solute occurs while the materials are in the fluid state when slow cooling is employed. When rapid freezing techniques are used special considerations forthcoming are in the dehydration step. These problems result from the fact that the dehydration must take place (the water must be removed) in the solid rather than the liquid state, i.e. sublimation.
In the prior art, the freeze substitution approach has involved the removal of tissue water by substituting a solvent or solvent-fixative mixture for the water at -75 to -80 degrees C. This introduces less severe solvent phase separation and chemical alteration artifacts to a tissue sample than past chemical fixation methodologies. From a practical standpoint freeze drying is complicated by the requirement that the tissue sample be warmed so as to increase the vapor pressure of the supercooled water and allow sublimation to proceed in a reasonable period of time. The increased temperature, in addition to increasing vapor pressure can produce a series of physical events leading to the expansion of ice crystals and concomitant damage to the ultrastructural morphology of the tissue sample. Many of the physical events which occur during the warming process have to do with transitions in the physical state of the water which is present. Changes which are typically encountered are glass transition, devitrification and recrystallization with an ensuing series of crystal lattice configuration transitions.
Thus it can be appreciated that freeze drying and other cryopreparation techniques present an exceptional opportunity for the preparation of tissue samples for morphological examination. However, inherent in the use of cryopreparation techniques are problems associated with dehydration and fixation of samples. These are the problems which are addressed by the process and apparatus of this invention.
The cryopreparation process of this invention has also demonstrated an extraordinary application in the transplanting of corneal tissue. Prior to this invention attempts to transplant corneas which involved a necessary freezing or freeze-drying of the corneas after removal from the donor invariably resulted in a clouded cornea upon transplanting. This physical condition of the transplanted cornea was caused by crystal formation in the cornea itself and concommitant damage to the stroma. Use of the process of this invention has enabled ophthalmologists to cryoprepare corneas immediately after removal from donors and to then transplant those corneas to the recipients with almost no clouding or crystal formation whatsoever. The ability to so transplant corneas represents an exceptional advantage to the process of this invention as well as a medical breakthrough in corneal transplant surgery.
It is therefore an object of this invention to provide a method for the preparation of biological samples without overt disruption or destruction of the morphological characteristics of the ultrastructure of the tissue sample.
It is a further object of this invention to provide a method for the preparation of tissue samples obtained in the solid vitreous phase by rapid freezing, the freezing process not resulting in unnecessary artifacts which restrict the interpretion by conventional analytical apparatus.
It is a still further object of this invention to provide a method for the preparation of tissue samples which permits the effective dehydration of the samples without corresponding damage to the tissue ultrastructure and which results in samples which can be used with modern high-powered magnification apparatus.
It is an additional object of this invention to provide a sample holder for use in the process of this invention.
These and other objects of this invention will be recognized from the description of the preferred embodiments which follows.